Hydrolysis of hydrocortisone acetate and delta1-hydrocortisone acetate



United States Patent Ofiice HYDROLYSIS, OF HYDROCORTISONE ACETATE ANDN-HYDROCORTISONE ACETATE Arthur R. Hanze, Kalamazoo Township, KalamazooCounty, Mich, assignor to The Upjohn Company, Kalamazoo, Mich, acorporation of Michigan No Drawing. Application June 10, 1955 Serial No.514,760

3 Claims. (Cl. 260- 3s7.4s

This invention relates to a process for the production of hydrocortisoneand n hydrocortisone, more particularly to an advantageous method forthe hydrolysis of hydrocortisone acetate and d -hydrocortisone' acetate.This application is a continuation-impart t my application- Ser; No.374,103, filed August 13, 1953, and now abandoned.

It is an object of the present invention to provide a process for thehydrolysis of hydrocortisone acetate and A -hydrocortisone acetate toproduce hydrocortisone and A hydr-ocortisone, respectively, in highyield. Other objects will be apparent to those skilled in the art towhich this invention pertains.

According to the present invention, hydrocortisone acetate or zt-hydrocortisone acetate dissolved in a substantially oxygen-freelower-alkanol is mixed with at least a molar equivalent of analkali-metal bicarbonate and the reaction is continued, in the absenceof atmospheric oxygen and significant amounts of carbon dioxide, at atemperature bet-ween about ten and about thirty degrees cen tigrade toproduce hydrocontisone which is then isolated from the reaction mixtureafter neutralizing the mixing with a dilute aqueous acid, preferably ata temperature below about thirty degrees centigrade.

The process of the present invention may be illustratively representedby the following formulae:

CH3 (I? CHr-O-C-CH3 7 alkali-metal O bicarbonate C|H9OH 0 0 l---0Hwherein the dotted line represents a double bond at the 1(2) positionwhich is present When the starting steroid is A -hydrocortisone acetate(11p,17a-dihydroxy-2l=ace toxyl ,4-pregnadiene-3,20-dione) Whenfollowing the method of the present. invention, substantially purehydrocortisone, usually in its low melting form, and A hydrocortisonecan be obtained and isolated in very high yield from the hydrolysis ofhydrocortisone acetate and A -hydrocortisone acetate, respec- PatentedJuly 8, 1958 tively, without the necessity of further purification. Asis well known in chemistry substantially quantitative yields of pureproduct, directly from areaction, are seldom obtained. This isparticularly true in reactions involving steroids having the labilellfi-hy-droxy group and/or the dihydroxy acetone side chain. However,when following the specific hydrolysis procedure of the presentinvention, about a 95 percent yield of pure hydrocor-tisone or n-hydrocortisone can be obtained from its acetate without any furtherpurification.

The prior art has hydrolyzed hydrocortisone acetate and other acetateesters of 21-hydroxy steroids. The use of both weak and strong alkalihas been suggested by Reichstein, U. S. 2,401,775. He didnot,.however,em-

ploy a means for maintaining the reaction mixture free from atmosphericoxygen and consequently, arlow yield of free alcohol was obtained. Thehydrolysis ofsteroids possessing the A -3-keto-11fi-hydroxy nucleus to.produce a steroid possessing a dihydroxy acetone side chain presentsunique problems. Minl-on and Tishler, U. S. 2,634,277, mention thatunsatisfactory results are obtained using acid or weakly alkalinehydrolyzing agents which give poor yields of free alcohol in thehydrolysis of A 3,1l,20 triketo-l7oc-hydroxy-2l-acyloxy-pregnane, i. e.,esters of cortisone, and required about two days for the hydrolysisoperation.

it has now been found that under the operating con ditions describedbelow, about a percent yield of hydrocortisone or tt -hydrocortisone canbe obtained from its acetate in about four hours, a result in directopposition to that described by Minlon and Tishler when using a relatedprocess on a related steroid. The use of an alkali metal bicarbonate asthe hydrolyzing agent, under the reaction conditions of the presentinvention, permits the use of less exact reaction conditions from thoseemployed 'by Min'lon and Tishler who employ precisely measuredequivalents of reactants and carefully controlled reaction times, whichconditions are difiicult to duplicate on an industrial scale, whilestill permitting a practically quantitative recovery of pure hydrolyzedsteroid.

The hydrolysis agents utilized herein are the alkalimetal bicarbonates,i. e., sodium bicarbonate, potassium bicarbonate and lithiumbicarbonate, with potassium bicarbonate or sodium bicarbonate beingpreferred and potassium bicarbonate being particularly desirable becauseof its greater solubility in the reaction mixture. A mole of alkalimetal bicarbonate per mole of starting steroid is theoretically requiredbut a substantial molar equivalent excess is usually used since optimumyields of the steroida1 free alcohol are thus obtained.

The starting hydrocortisone acetate or n -hydrocortisone acetate isdissolved in a lower-alkanol, e. g., methanol, ethanol, isopropanol,tertiary butyl alcohol, preferably methanol, and mixed with thealkali-metal bicarbonate dissolved in Water. Since free oxygen in thereaction mixture is disadvantageous, the reaction is carried out in thesubstantial absence of oxygen. This is preferably achieved by bubblingnitrogen through the methanol and the aqueous solutions prior to theirmixing and then maintaining the mixture in a nitrogen atmosphere byflushing oxygen free nitrogen over the surface and/or by bubbling itthrough the mixture during the reaction. Antioxidants may advantageouslybe employed to inhibit oxidation reactions.

The reaction is carried out at between about ten and about thirtydegrees centigrade. Since the reaction is exothermic, the reactiontemperature can conveniently be maintained within these limits bycooling the methanol and aqueous solutions to about ten to twentydegrees centigrade prior to their mixing. During the reaction, thetemperature will then rise to about room temperature if no externalheating or cooling is applied. Although the reaction is substantiallycomplete in a relatively short time, the reaction mixture is preferablymaintained for several hours to ensure a complete reaction.

' The thus-produced hydrocortisone or n -hydrocortisone is then isolatedby neutralizing the reaction mixture with 'an'acid, preferably a weakacid such as, for example, acetic acid, and preferably in the cold. Forcomplete precipitation of the hydrocortisone, the neutralized solutionis concentrated by distillation of the organic solvent therefrom atreduced pressure, preferably at or below room temperature. A furtherprocedure to ensure complete precipitation of the hydrocortisone or A-hydrocortisone involves chilling the thus-concentrated solution.Filtering the concentrated and chilled solution separates substantiallyall of the steroidal material from the mixture. Washing the filter cakewith cold water followed by drying is productive of from about ninetypercent to an almost quantative yield of substantially purehydrocortisone or A hydrocortisone.

The following examples are illustrative of the process and products ofthe present invention which is not to 'be construed as limited thereto.

PREPARATION l 2,21 diethoxyoxalyl 11 ketoprogesterone and sodiumdienolate thereof Nineteen milliliters (0.136 mole) of ethyl oxalate and21.2 milliliters (0.047 mole) of a 2.2 Normal methanolic solution ofsodium methoxide was added to a solution of 6.9 grams (0.021 mole) ofll-ketoprogesterone in 100 milliliters of anhydrous tertiary butylalcohol at about fifty degrees centigrade. The mixture was maintained atroom temperature for three hours, whereafter the precipitated sodiumdienolate of 2,21-diethoxyoxalyl-11- ketoprogesterone was filtered,washed with ether and then dissolved in water. The aqueous solution wasacidified with dilute hydrochloric acid and the thus-precipitated2,21-diethoxyoxalyl-11=ketoprogesterone was filtered therefrom and thendried to yield 10.2 grams, a yield of 92 percent of the theoretical, of2,21-diethoxyoxalyl-ll-ketoprogesterone in the form of a, yellowamorphous powder which exhibited a reddish color in an alcoholic ferricchloride solution.

PREPARATION 2 Methyl 2 bromo 3,11 diketo 4,I7(20) pregnadiene-ZI -ate Asolution of eight grams (0.015 mole) of the 2,21-diethoxyoxalyl-ll-ketoprogesterone obtained according to the methoddescribed in Preparation 1 and 5.9 grams (0. 60 mole) of anhydrouspotassium acetate in 140 milliliters of methanol was cooled to zerodegrees centigrade in an ice bath and a solution of 7.4 grams (0.046mole) of bromine in 74 milliliters of methanol was then added dropwisethereto over a period of about one-half hour to produce2,21,21-tribromo-2,21-diethoxyoxalyl-1I-ketoprogesterone. To theresulting mixture was then added about fifty milligrams of phenol and 67milliliters (0.100 mole) of a 1.5 Normal methanolic solution of sodiummethoxide whereafter the mixture was heated for five minutes on a steambath followed by the addition of the cooled solution to water. Aflocculent white precipitate of methyl2-bromo-3,1l-diketo-4,17(20)-pregnadiene-21- oate formed, and, afterbeing thoroughly washed with water and dried in a vacuum desiccator,weighed 6.77 grams and melted at 74 to 94 degrees centigrade. 1.50 gramsof this impure product was chromatographed over 150 grams of Florisilmagnesium silicate. The column was developed with ZOO-milliliterportions of solvents of the following composition and order: one ofbenzene, ten of Skellysolve B hexane hydrocarbons plus five percentacetone, and ten of Skellysolve B plus 7.5 percent acetone. The second,third and fourth portions grade.

of Skellysolve B plus 7.5 percent acetone eluates were combined and theproduct was distilled therefrom leaving 382 milligrams of productmelting at to 154 degrees centigrade. Recrystallization of thesecrystals from methanol gave analytically pure methyl2-bromo-3,1l-diketo- 4,17(20)-pregnadiene-21-oate as transparent prismswhose melting point varied between 155 to degrees centigrade and 160 to162 degrees centigrade, depending upon the rate of heating.

PREPAMTION 3 Methyl 3 -ket0-1 ,4,1 7'( 20) -pregnatriene-21 -0ate Amixture of 0.21 gram of methyl 2-bromo-3-keto-4,l7(20)-pregnadiene-2l-oate and 0.8 milliliter of redistilled-collidine was heated at the refluxing temperature of the mixture forthirty minutes and then cooled to room temperature. The cooled mixturewas diluted with ether and the 29 milligrams of collidine hydrobromidewhich precipitated was filtered from the solution. The filtrate waswashed with dilute sulfuric acid followed by water and then dried overanhydrous sodium sulfate. The dried solution was freed of solvent andthe residue was crystallized from ethyl acetate to give methyl 3-keto-1,4,17(20)-pregnatriene-2l-oate melting at 209.2 to 218.7 degreescentigrade and, after recrystallization from ethyl acetate, melting at229.2 to 233 degrees centigrade.

PREPARATION 4 11/3,2]-dihydr0xy-1,4,I 7(20 -pregnatriene-3-one Asolution of 1.0 gram of 3,11-diketo-1,4,17(20)- pregnatriene-Zl-oic acidmethyl ester, 0.5 milliliter of pyrrolidine, and 1.5 milligrams ofpara-toluenesulfonic acid in 100 milliliters of benzene were heated atthe reflux temperature of the mixture for six hours with the concomitantremoval of the water of reaction. The solvent was distilled from themixture to leave a residue of3-pyrrolidyl-1,3,5,l7(20)-pregnatetraene-21-oic acid methyl ester.

This residue was dissolved in five milliliters of benzene, a suspensionof 376 milligrams of lithium aluminum hydride in 290 milliliters ofether was added thereto over a period of five minutes, and the mixturewas stirred at room temperature for one hour. Three milliliters of ethylacetate were added to destroy excess lithium aluminum hydride, followedby five milliliters of water. The ether was distilled at atmosphericpressure from the mixcure to leave a residue of3-pyrrolidyl-1113,21-dihydroxy- 1,3,5,17(20)-pregnatetraene andinorganic material.

This crude distillation residue was mixed with 327 milliliters ofmethanol at 55 degrees centigrade until solution was effected and thencooled to 37 degrees centigrade. 116.5 milliliters of an aqueous fivepercent sodium hydroxide solution was added thereto and heating of themixture at about forty degrees centigrade was continued for ten minutes.The solution was neutralized with 3.5 milliliters of acetic acid atbelow 37 degrees centigrade and the solvent distilled therefrom atreduced pressure and at a temperature below 45 degrees centi- The 140milliliters of residue was mixed with a mixture of 370 milliliters ofwater and milliliters of concentrated sulfuric acid, stirred for twentyminutes and then filtered and washed with water to give 113,21-dihydroxy-1,4,17(20)-pregnatriene-3-one which melts at 149 to 153degrees centigrade when recrystallized from ethylene dichloride, has an[a] of plus 117 degrees in chloroform, and an 6 of 14,700. The meltingpoint varies considerably, depending upon the crystallizing solvent.

PREPARATION 5 11B hydroxy 21 acetoxy 1,4,17(20)-pregnatriene 3-0ne Asolution of 6.6 grams of 11B,21-dihydroxy-1,4,

17(20)-pregnatriene-3-one in fifteen milliliters of pyridine, preparedat room temperature, was mixed, at zero degrees centigrade, with fifteenmilliliters of acetic anhydride. Aften one hour at room temperature,copious crystallization had taken place in the resulting mixture. Afterstirring for a total of eighteen hours, the mixture was filtered and thecake washed with a 50:50 mixture of pyridine and acetic anhydride,followed by water. The'cake Wasdried to give 4.92 grams of llp-hydroxy-21-acet0xy-1,4,17(20)-pregnatriene-3-one melting at 219 to 223 degreescentigrade. Mixing the original filtrate with water gave 2.12 grams ofless pure 11p-hydroxy-21- acetoxy-1,4,17(20)-pregnatriene-3-one meltingat 186 to 207 degrees centigrade.

PREPARATION 6 1119,1711 dihydroxy 21 acetoxy 1,4 pregnadiene 3,20 dioneTo a solution of 1.11 grams (3 millimoles) of 11,8- hydroxy 21 acetoxy1,4,l7(20) pregnatriene 3 one in fifty milliliters of tertiary butylalcohol was added 1.5 milliliters of pyridine followed by 5.02milliliters of a tertiary butyl alcohol solution of 7.5 millimoles of N-'methylmorpholine oxide peroxide (prepared by the reac tion of 7.5millimoles of N-methylmorpholine with 15.0 millimoles of anhydroushydrogen peroxide in the tertiary butyl alcohol) followed by 18.4milligrams of osmium tetroxide in ten milliliters of tertiary butylalcohol. The solution, which, within five minutes had turned orange-red,was maintained for ninety minutes at 25 degrees centigrade. At the endof this time, the now straw colored solution was mixed with 23milliliters of 0.5 percent aqueous sodium sulfite at room temperaturefor 25 minutes and then concentrated, at reduced pressure, to a volumeof about forty milliliters. This concentrate was stirred for thirtyminutes and 35 milliliters of water was then added portionwise over aperiod of fifteen minutes. After stirring for 45 minutes, the solutionwhich had gradually precipitated crystals, was filtered, washed with a1:3 mixture of tertiary butyl alcohol and water, and dried to give 670milligrams of 11,8,17u-dihydroxy- 21-ace'toxy-1,4-pregnadiene-3,20-dionewhich melted at 239 to 245 degrees centigrade. Recrystallization ofthese crystals from hot acetone raised the melting point to 240 to 242degrees centigrade. The filtrate yielded crystals of 11,8,1706 dihydroxy21 acetoxy 1,4 pregnadiene- 3,20-dione which, after two crystallizationsfrom hot acetone, melted at 243 to 247 degrees centigrade.

EXAMPLE 1 1],B,]7a,21 trihydroxy 4 pregnene 3,20 dione (hydrocortisone)A solution of 4.04 grams (ten millimoles) of hydrocortisone acetate inmethanol was freed of oxygen gas by bubbling nitrogen therethrough. Asolution of 4.04 grams (40.4 millimoles) of potassium bicarbonate inforty milliliters of water was similarly freed of oxygen. The twosolutions were mixed at a temperature of between eighteen and twentydegrees centigrade and in a nitrogen atmosphere. The temperature of thesolution rose to between 24 and 26 degrees centigrade and some of thepotassium bicarbonate precipitated from the solution. The mixture wasstirred at room temperature for five hours while protecting it fromatmospheric oxygen with nitrogen. During the first ninety minutes ofstirring the solution became homogeneous. At the end of five hours thesolution was neutralized by the addition of sixty milliliters of icewater containing three milliliters of glacial acetic acid. Theneutralized solution was concentrated to about sixty milliliters bydistillation at room temperature at reduced pressure and then chilled ina refrigerator for about sixteen hours. The thus-precipitatedhydrocortisone was filtered, washed with ice-water and then dried toyield 3.455 grams, a yield of 95.3 percent of the theoretical, of thelow-melting form of hydrocortisone melting at 202 to 205 degreescentigrade, having an sesame [111 of plus 167 degrees (in percentethanol) and an E of 15,725. Papergram analysis substantiated the purityof the product.

Similar results can be obtained by substituting a molar equivalent ofsodium bicarbonate or lithium bicarbonate for the potassium bicarbonateused in the reaction.

EXAMPLE 2 11/3,17ot,21 trihydroxy 1,4 pregnadiene 3,20 dione A solutionof seven grams of11,8,17a-dihydroxy-2lacetoxy-1,4-pregnadiene-3,20-dione in 700milliliters of methanol was freed of oxygen by bubbling nitrogentherethrough. A solution of seven grams potassium bicarbonate in seventymilliliters of water was similarly freed of oxygen and carbon dioxide.The two solutions were mixed and then stirred in an atmosphere ofnitrogen, produced by flushing nitrogen over the mixture, for fourhours. To the solution was then added 5.25 milliliters of glacial aceticacid in milliliters of water and stirring was continued whileconcentrating the mixture by distillation of the methanol at reducedpressure until copious precipitation occurred. The 200 milliliters ofconcentrated mixture was chilled to about zero degrees centigrade,filtered and the filter cake washed well with ice water. The driedfilter cake consisted of 5.64 grams, a yield of 89.9 percent of thetheoretical, of substantially pure11,8,17a,2l-trihydroxy-1,4-pregnadiene-3,20- dione melting at 225 to 230degrees centigrade with de composition, having an [a] of plus 98 degreesin dioxane and an E of 14,100. The filtrate was concentrated at reducedpressure to about milliliters. The resulting precipitate was filteredand dried to yield an additional 0.52 gram, a yield of 8.3 percent ofthe theoretical, of 1 1,8,17et,2l-trihydroxy-1,4-pregnadiene-3,20-dionemelting at 224 to 239 degrees centigrade.

It is to be understood that the invention is not to be limited to theexact details of operation described, as obvious modifications andequivalents will be apparent to one skilled in the art, and theinvention is therefore to be limited only by the scope of the appendedclaims.

I claim:

1. The process which comprises hydrolyzing a compound selected from thegroup consisting of hydrocortisone acetate and Al -hydrocortisoneacetate with at least a molar equivalent, calculated on the startingsteroid, of an alkali metal bicarbonate in a substantially oxygen-freesolution of a mixture of lower-alkanol and water and continuing thereaction at a temperature between about ten and about thirty degreescentigrade while protecting the mixture from atmospheric oxygen; second,neutralizing the saponified mixture with acid; and third, isolating thethus-produced hydrolyzed steroid.

2. A process for the production of substantially pure hydrocortisonewhich comprises the steps of first, hydrolyzing hydrocortisone acetateby mixing a substantially oxygen-free solution of11B,17a-dihydroxy-21-acetoxy-4- pregnene-3,20-dione in methanol with anaqueous, substantially oxygen-free solution of potassium bicarbonate inexcess of a molar equivalent, calculated on the hydrocortisone acetate,at a temperature between about ten and about thirty degrees centigrade,and continuing the reaction in a substantially oxygen-free, nitrogenatmosphere, at a temperature between about ten and about thirty degreescentigrade; second, neutralizing the hydrolyzed mixture, at atemperature below about thirty degrees centigrade with acetic acid; andthird, separating the thus-produced hydrocortisone from the resultingmixture.

3. A process for the production of substantially pure A hydrocortisonewhich comprises the steps of first, hydrolyzing A -hydrocortisoneacetate by mixing a substantially oxygen-free solution of115,17a-dihydroxy-21- acetoxy-1,4-pregnadiene-3,20-dione in methanolwith an aqueous, substantially oxygen-free solution of potassiumbicarbonate in excess of a molar equivalent, cal- 7 8 culated on the A-hydrocortisone acetate, at a temperature separating the thus-produced A-hydrocortisone from the between about ten and about thirty degreescentigrade, resulting mixture. and continuing the reaction in asubstantially oxygen-free, References Cited in the file of this' patentnitrogen atmosphere, at a temperature between about ten and about thirtydegrees centigrade; second, neutralizing 5 UNITED STATES PATENTS thehydrolyzed mixture at a temperature below about 2,401,775 ReichsteinJune 11, 1946 thirty degrees centigrade with acetic acid; and third,2,634,277 Minlon Apr. 7, 1953

1. THE PROCESS WHICH COMPRISES HYDROLYZING A COMPOUND SELECTED FROM THEGROUP CONSISTING OF HYDROCORTISONE ACETATE AND $1-HYDROCORTISONE ACETATEWITH AT LEAST A MOLAR EQUIVALENT, CALCULATED ON THE STARTING STEROID, OFAN ALKALI-METAL BICARBONATE IN A SUBSTANTIALLY OXYGEN-FREE SOLUTION OF AMIXTURE OF LOWER-ALKANOL AND WATER AND CONTINUING THE REACTION AT ATEMPERATURE BETWEEN ABOUT TEN AND ABOUT THIRTY DEGREES CENTIGRADE WHILEPROTECTING THE MIXTURE FROM ATMOSPHERIC OXYGEN; SECOND, NEUTRALIZING THESAPONIFIED MIXTURE WITH ACID; AND THIRD, ISOLATING THE THUS-PRODUCEDHYDROLYZED STEROID.